Progesterone (P4) is an essential hormone that regulates the entire reproductive process. P4 has many target sites throughout the reproductive axis but two major sites of action include the ovary and the uterus. The ovary synthesizes and secretes P4 throughout the menstrual/estrous cycle with its levels increasing during pregnancy. P4 acts directly of the granulosa cells of the ovary to inhibit mitosis and apoptosis. In addition, the P4 promotes the viability and steroidogenic potential of luteal cells and stimulates both its own secretion and cholesterol synthesis. Many of the actions of P4 within the ovary and uterus are mediated by classical nuclear P4 receptors (PGRs). However, not all of the actions of P4 can be explained by activation of PGRs, since a number of cell lines that do not express these receptors, as well as Pgr null mice, are able to respond to P4. Our recent in vitro studies of ovarian and uterine cells have revealed that some actions of P4 such as granulosa/luteal cell viability, P4 synthesis and uterine stromal cell differentiation are mediated in part through the P4 binding protein, Progesterone Receptor Membrane Component-1 (PGRMC1). Based on these in vitro data, we hypothesize that PGRMC1 plays essential roles in regulating ovarian and uterine function in vivo. In this grant we will directly test our hypothesis by generating a conditional knockout mouse in which PGRMC1 is specifically depleted within the granulosa/luteal cells of the ovary and the mesenchymal (stromal) tissue of the uterus (Specific Aim 1). We will then use the conditional PGRMC1 knockout mouse to monitor the effects of depleting PGRMC1 on 1) female fertility, 2) ovarian function (i.e. follicular growth, steroid synthesis and ovulation) and 3) uterine function (i.e. endometrial decidualization and embryo implantation) (Specific Aim 2). In Specific Aim 3 we will isolate PGRMC1 binding proteins by immunoprecipitation using lysates of ovarian and uterine tissues and then using two- dimensional gel electrophoresis and subsequent proteomics to identify PGRMC1 binding partners. The successful completion of the proposed studies will provide compelling evident to support PGRMC1's role in female reproductive physiology. What is important about establishing PGRMC1 as a mediator of specific ovarian and uterine functions is that this will allow for the development of a new class of P4 antagonists and agonists. These putative PGRMC1 modulators would be completely different from the present-day PGR antagonists and would only interfere with the actions of P4 that are mediated via PGRMC1 and not the PGR. These putative PGRMC1 modulators could find application in the treatment of human fertility and potentially ovarian cancers, since PGRMC1 promotes the viability of these cancers. PUBLIC HEALTH RELEVANCE: Progesterone (P4) is an essential hormone that regulates the entire female reproductive process. Our recent in vitro studies of ovarian and uterine cells have revealed that some actions of P4 such as granulosa/luteal cell viability, P4 synthesis and uterine stromal cell differentiation are mediated in part through the P4 binding protein, Progesterone Receptor Membrane Component-1 (PGRMC1). In this grant we will conclusively determine the role of PGRMC1 by generating a conditional knockout mouse and then monitoring the effects of depleting PGRMC1 on 1) female fertility, 2) ovarian function and 3) uterine function. If successful, the proposed studies could lead to the development of a new class of P4 antagonists and agonists that could be used to treat human infertility and potentially ovarian and uterine cancers.